A gene construct comprising a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, organisms which comprise this gene construct, the use of these sequences or of the gene construct for preparing biotin, and a process for preparing biotin are described.
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1. A process for preparing biotin, which comprises expressing a biotin synthase gene selected from the group consisting of sequences SEQ ID NO: 1 and SEQ ID NO: 3 in a prokaryotic or eukaryotic host organism able to synthesize dethiobiotin.
5. A gene construct comprising a biotin synthase gene selected from the group of sequences consisting of SEQ ID NO:1 and SEQ ID NO:3, which is functionally linked to sequences selected from the group consisting of one or more heterologous regulatory sequences and a genetically modified natural regulatory sequence, where the natural regulation by biotin has been switched off.
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7. An organism selected from the group consisting of bacteria, fungi, yeasts and plants comprising a gene construct as claimed in
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This application is a 371 of International Application No. PCT/EP98/04097 published as WO 99/05285 which has an international filing date of Jul. 2, 1998.
The invention relates to a gene construct comprising a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, to organisms which comprise this gene construct, to the use of these sequences or of the gene construct for preparing biotin, and to a process for preparing biotin.
Biotin (vitamin H) plays an essential role as coenzyme in enzyme-catalyzed carboxylation and decarboxylation reactions. Biotin is thus an essential factor in living cells. Almost all animals and some microorganisms have to take biotin in from the outside because they are unable to synthesize biotin themselves. It is thus an essential vitamin for these organisms. Bacteria, yeasts and plants by contrast are themselves able to synthesize biotin from precursors (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5).
Biotin synthesis has been investigated in bacterial organisms, specifically in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus sphaericus (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326). The first intermediate known to date in E. coli is regarded as being pimelyl-CoA (Pm-CoA), which derives from fatty acid synthesis (DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5 1996). The synthetic pathway for this biotin precursor in E. coli is substantially unknown at present (Ifuku 1993, Lemoine 1996). Two genes, bioC and bioH, whose corresponding proteins are responsible for the synthesis of Pm-CoA, have been identified. The enzymatic function of the gene products BioH and BioC is not known at present (Lemoine et al., Mol. Microbio. 19, 1996: 645-647, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5). Pm-CoA is converted into biotin in four further enzymatic steps. Starting from Pm-CoA there is initially condensation with alanine to give 7-keto-8-aminopelargonic acid (KAPA). The gene product for this conversion is BioF (KAPA synthetase). KAPA is transaminated by BioA (DAPA aminotransferase) with the cosubstrate S-adenosylmethionine to give 7,8-diaminopelargonic acid. The next step results, after an ATP-dependent carboxylation reaction, in dethiobiotin (DTB) and is catalyzed by BioD (dethiobiotin synthase). In the last step, DTB is converted into biotin. This step is catalyzed by BioB (biotin synthase). The genes bioF, bioA, bioD, and bioB coding for the proteins-which have been described are encoded in E. coli on a bidirectional operon. This operon is located between the λ attachment site and the uvrB gene locus at about 17 minutes on the E. coli chromosome (Berlyn et al. 1996: 1715-1902). Two other genes are additionally encoded on this operon, one of which, bioc, has functions which have already been described in the synthesis of Pm-CoA, while it has not yet been possible to assign a function to an open reading frame behind bioA (WO94/8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577-85). Highly conserved homologs to the E. coli proteins BioF, A, D, B have been found in B. sphaericus, B. subtilis, Syneccocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, Bower et al., J. Bacteriol. 175, 1996: 4122-4130, Kaneko et al., DNA Res. 3, 3, 1996: 109-136), archaebacteria such as Methanococcus janaschi, yeasts such as Saccharomyces cerevisiae (Zhang et al., Arch. Biochem. Biophys. 309, 1, 1994: 29-35) or in plants such as Arabidopsis thaliana (Baldet et al., C. R. Acad. Sci. III, Sci. Vie. 319, 2, 1996: 99-106)).
The synthesis of Pm-CoA appears to take place differently in the two Gram-positive microorganisms which have been investigated to date than in E. coli. It has not been possible to find any homologs of bioH and bioC (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326).
Biotin is an optically active substance with three centers of chirality. It is currently prepared commercially only in a multistage costly chemical synthesis.
As an alternative to this chemical synthesis, many attempts have been made to set up a fermentation process for preparing biotin using microorganisms. It has been possible, by cloning the biotin operon on multi-copy plasmids, to increase biotin synthesis in microorganisms transformed with these genes. A further increase in biotin synthesis has been achieved by deregulation of biotin gene expression via selection of birA mutants (Pai C. H., J. Bacteriol. 112, 1972: 1280-1287). Combining the two approaches, ie. expression of the plasmid-encoded biosynthesis genes in a regulation-deficient strain (EP-B-0 236 429), resulted in a further increase in productivity. In this case, either the biotin operon can remain under the control of its native bidirectional promoter (EP-B-0 236 429), or its genes can be placed under the control of a promoter which can be regulated externally (WO94/8023).
It has not been possible to achieve commercially adequate productivity by previous approaches to the preparation of biotin by fermentation in E. coli. It has emerged that the yield in the preparation of biotin by fermentation is caused by the incomplete conversion of DTB into biotin by the BioB gene product (biotin-synthase). Cells which harbor mutations in the bioB gene are unable to grow on DTB and thus to convert DTB into biotin. The chemical and enzymatic mechanism of the conversion of DTB into biotin is at present only incompletely understood and elucidated.
Intensive genetic investigations to date have been unable to identify further proteins involved in the reaction. Characterization of the conversion of DTB into biotin has hitherto been carried out only in bacterial and plant cell extracts (WO94/8023, EP-B-0 449 724, Sanyal et al. Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48-56 and Biochemistry 33, 1994: 3625-3631, Baldet et al. Europ. J. Biochem. 217, 1, 1993: 479-485, Méjean et al. Biochem. Biophys. Res. Commun., Vol. 217, No. 3, 1995: 1231-1237, Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 1738-1741).
These investigations have shown that low molecular weight factors such as S-adenosylmethionine, NADPH, cysteine, thiamine, Fe2+, asparagine, serine, fructose 1,6-bisphosphate stimulate the synthesis of biotin (Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 1738-1741, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuk et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185-189). Besides these low molecular weight factors, other proteins which stimulate the synthesis of biotin from DTB in the presence of BioB have been identified. These are flavodoxin and flavodoxin-NADPH reductase (Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185-189, Sanyal et al., Arch. Biochem. Biophys. 326, 1, 1996: 48-56).
The biotin synthesis and lipoic acid synthesis exhibit great homology. In both synthetic pathways there is insertion of a sulfur, or two sulfur atoms, between non-activated carbon atoms in the last stage of the synthesis. The synthesis of lipoic acid is at present only inadequately characterized (DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5). To date, only two necessary genes have been identified in E.coli: lipA and lipB. Both genes are located in an operon, with an as yet uncharacterized open reading frame (=ORF) between the two genes. Another gene lplA is able to transfer lipoic acid via a lipoyl-AMP intermediate to lysine. This reaction is thus similar to the activity of birA. Homologous regions in the amino acid sequence have been identified by sequence comparisons between LipA and BioB. These include, inter alia, a cysteine cluster. It has been shown that LipA catalyzes the incorporation of two sulfur atoms into the lipoic acid (DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5).
The results concerning the origin of the sulfur in the biotin molecule are contradictory. Investigations on biotin synthesis in whole cell extracts showed that radioactivity was incorporated into biotin in the presence of 35S-labeled cysteine; sulfur incorporation into the biotin molecule was undetectable either with 35S-labeled methionine or with S-adenosylmethionine (Ifuku et al., Biosci. Biotechnol. Biochem. 59, 2, 1995: 184-189, Birch et al., J. Biol. Chem.270, 32, 1995: 19158-19165).
This contrasts with investigations on purified BioB protein in the presence of 35S-labeled cysteine and without addition of cell extracts, in which case although biotin synthesis was observed there was no incorporation of radioactivity into the biotin molecule (Sanyal et al., Arch. Biochem. Biophys. 326, 1, 1996: 48-56, Méjean et al., Biochem. Biophys. Res. Commun. 2127, 3, 1995: 1231-1237). Under these synthesis conditions, without addition of cell extract, the amount of biotin formed was small and corresponded to a maximum of about 2 mol of biotin/mol of BioB (Sanyal et al., Arch. Biochem. Biophys. 326, 1, 1996: 48-56) or 0.1 mol of biotin/mol of BioB (Méjean et al., Biochem. Biophys. Res. Commun. 2127, 3, 1995: 1231-1237). According to these investigations, sulfur can be incorporated into biotin without using cysteine as sulfur donor. This biotin formation without an external source of sulfur might be explained by a transfer of sulfur from the 2Fe-2S cluster which has been detected in BioB. The actual source of sulfur for biotin synthesis is still unclear. It has thus not yet been possible to demonstrate a genuine catalytic activity of BioB in vitro.
Despite this large number of approaches, the yield of biotin from microbial fermentation is currently insufficient for industrial production.
It is an object of the present invention to develop an industrial process for preparing biotin by fermentation which optimizes as far as possible the conversion of dethiobiotin into biotin, and thus makes improved biotin synthesis possible.
We have found that this object is achieved by the novel process for preparing biotin which comprises expressing a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, and its functional variants, analogs or derivatives, in a prokaryotic or eukaryotic host organism able to synthesize biotin, cultivating this organism and using the synthesized biotin directly, after removal of the biomass or after purification of the biotin.
The biotin genes used in the novel process and having the sequences SEQ ID No. 1 and SEQ ID No. 3, respectively, are kept in the SwissProt databank under accession numbers AE000364 and D90811. The sequence D90811 has additionally been described by Aiba et al. in DNA Res. 3, 6, 1996: 363-377. Homology with the NifS protein has been noted for both sequences in the databank. Further information on these sequences is not available from the databank or the publication.
The productivity of biotin synthesis can be markedly increased by expressing the sequences SEQ ID No. 1 and/or SEQ ID No. 3 in a prokaryotic or eukaryotic host organism. Expression of the genes increases the synthesis of biotin from dethiobiotin by a factor of at least 2 by comparison with the control without these genes, preferably by a factor of more than 3. The sequence SEQ ID No. 1 is preferably used.
After isolation and sequencing it is possible to obtain the biotin genes which are used in the novel process, have the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 3, and code for the amino acid sequences indicated in SEQ ID No: 2 and SEQ ID No. 4, or their allelic variants. Allelic, variants mean variants of SEQ ID No. 1 or SEQ ID No. 3 displaying from 40 to 100% homology at the amino acid level, preferably from 50 to 100%, very particularly preferably from 80 to 100%. Allelic variants comprise, in particular, functional variants obtainable by deletion, insertion or substitution of nucleotides from the sequence depicted in SEQ ID No: 1 or SEQ ID No: 3, where the enzymatic activity is, however, retained.
Analogs of SEQ ID No: 1 or SEQ ID No: 3 mean, for example, their bacterial, fungal, plant or yeast homologs, truncated sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence.
Derivatives mean, for example, promoter variants. The promoters which precede the stated nucleotide sequences can be altered by one or more nucleotide exchanges, by insertion(s) and/or deletion(s) without, however, adversely affecting the functionality or activity of the promoters. The promoters can furthermore have their activity increased by altering their sequence, or be replaced completely by more effective promoters, even from heterologous organisms.
Derivatives also mean variants whose nucleotide sequence has been altered in the region from -1 to -30 in front of the start codon in such a way that gene expression and/or protein expression is increased. This is advantageously effected by altering the Shine-Dalgarno sequence.
Suitable prokaryotic host organisms in the novel process are, in principle, all biotin-synthesized Gram-negative or Gram-positive bacteria. Examples which may be mentioned of Gram-negative bacteria are Enterobacteriaceae such as the genera Escherichia, Aerobacter, Enterobacter, Citrobacter, Shigella, Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonadaceae such as the genera Pseudomonas, Xanthomonas, Burkholderia, Gluconobacter, Nitrosomonas, Nitrobacter, Methanomonas, Comamonas, Cellulomonas or Acetobacter, Azotobacteraceae such as the genera Azotobacter, Azomonas, Beijerinckia or Derxia, Neisseriaceae such as the genera Moraxella, Acinetobacter, Kingella, Neisseria or Branhamella, the Rhizobiaceae such as the genera Rhizobium or Agrobacterium or the Gram-negative genera Zymomonas, Chromobacterium or Flavobacterium. Examples of Gram-positive bacteria which may be mentioned are the endospore-forming Gram-positive aerobic or anaerobic bacteria such as the genera Bacillus, Sporolactobacillus or Clostridium, the coryneform bacteria such as the genera Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium, Microbacterium or Kurthia, the Actinomycetales such as the genera Mycobacterium, Rhodococcus, Streptomyces or Nocardia, the Lactobacillaceae such as the genera Lactobacillus or Lactococcus, the Gram-positive cocci such as the genera Micrococcus or Staphylococcus.
Bacteria preferably used in the novel process are of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium or Staphylococcus. Particularly preferred genera and species are Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas mutabilis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Comamonas acidovorans, Comamonas testosteroni, Acinetobacter calcoaceticus, Azotobacter vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Arthrobacter citreus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium sp., Brevibacterium ketoglutamicum, Brevibacterium linens, Brevibacterium sp., Streptomyces lividans, Rhizobium leguminosarum or Agrobacterium tumefaciens. Bacteria which already have increased natural biotin production are advantageously used.
The taxonomic positioning of the stated genera has been subject to great change in recent years and is still in a state of flux because incorrect genus and species names are being corrected. This frequent need in the past for taxonomic regrouping of said genera within the classification of bacteria means that families, genera and species other than those mentioned above are also suitable for the novel process.
Suitable eukaryotic host organisms for the novel process are, in principle, all biotin-synthesizing organisms such as fungi, yeasts, plants or plant cells. Yeasts which may be mentioned as preferred are the genera Rhodotorula, Yarrowia, Sporobolomyces, Saccharomyces or Schizosaccharomyces. Particularly preferred genera and species are Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor, Sporobolomyces shibatanus or Saccharomyces cerevisiae.
It is possible in principle to use all plants as host organism, and preferred plants are those important in livestock feeding or human nutrition, such as corn, wheat, barley, rye, potatoes, peas, beans, sunflowers, palms, millet, sesame, copra or oilseed rape. Plants such as Arabidopsis thaliana or Lavendula vera are also suitable. Plant cell cultures, protoplasts from plants or callus cultures are particularly preferred.
It is advantageous to use in the novel process microorganisms such as bacteria, fungi, yeasts or plant cells which are able to secrete biotin into the culture medium and which, where appropriate, additionally have increased natural biotin synthesis. It is also possible and advantageous for these organisms to be defective in respect of the regulation of their biotin biosynthesis, ie. there is no, or only very diminished, regulation of the synthesis. This regulation defect results in these organisms having a considerably higher biotin productivity. A regulation defect of this type is known, for example, for Escherichia coli as the birA defective mutant and ought preferably to be present in the cells in the form of a defect which can be inducted by external influences, for example is temperature-inducible. Organisms which do not naturally produce biotin can also be used in principle after they have been transformed with the biotin genes.
In order to increase the overall biotin productivity further, the organisms in the novel process advantageously ought additionally to comprise at least one other biotin gene selected from the group of bioA, bioB, bioF, bioC, bioD, bioH, biop, biow, bioX, bioy or bioR. This additional gene or these additional genes may be present in one or more copies in the cell. They may be located on the same vector as the sequences SEQ ID No. 1 and/or SEQ ID No. 3, or have been integrated on separate vectors or else into the chromosome. The sequences SEQ ID No. 1 and/or SEQ ID No. 3 can also be inserted into the genome.
The novel gene construct means the biotin gene sequences SEQ ID No. 1 and SEQ ID No. 3 and their functional variants, analogs or derivatives, which are functionally linked to one or more regulatory signals to increase gene expression. In addition to these novel regulatory sequences, the natural regulation of these sequences in front of the actual structural genes may still be present and, where appropriate, have been genetically modified so that the natural regulation has been switched off and the expression of the genes has been increased. However, the gene construct may also have a simpler structure, ie. no additional regulatory signals have been inserted in front of the sequences SEQ ID No. 1 and/or SEQ ID No. 3, and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequences have been mutated in such a way that there is no longer any regulation by biotin, and gene expression is increased. It is also possible to insert additional, advantageous regulatory elements at the 3' end of the DNA sequences. The biotin genes having sequences SEQ ID No. 1 and/or SEQ ID No. 3 may be present in one or more copies in the gene construct.
Advantageous regulatory sequences for the novel process are, for example, present in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq-, T7, T5, T3, gal, trc, ara, SP6, λ-PR or the λ-PL promoter, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, present in the Gram-positive promoters amy and SPO2, in the yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH or in the plant promoters CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
It is possible in principle for all natural promoters with their regulatory sequences like those mentioned above to be used for the novel process. It is furthermore possible and advantageous to use synthetic promoters.
The gene construct may comprise further biotin genes selected from the group of bioA, bioB, bioF, bioC, bioD, bioH, biop, biow, bioX, bioy or bioR in one or more copies, each of which may have its own promoter or else may be under the control of the promoter of sequences SEQ ID No. 1 or SEQ ID No. 3.
The gene construct is, for expression in the abovementioned host organism, advantageously inserted into a host-specific vector which makes optimal expression of the genes in the host possible. Examples of suitable vectors in E. coli are pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI, in Streptomyces are pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus are pUB110, pC194 or pBD214, in Corynebacterium are pSA77 or pAJ667, in fungi are PALS1, pIL2 or pBB116, in yeasts are YEp6, YEpl3 or pEMBLYe23 or in plants are pLGV23, pGHlac+, pBIN19, pAK2004 or pDH51. Said vectors represent a small selection from the possible vectors. Further vectors are well-known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985 , ISBN 0 444 904018).
Expression systems mean the combination of the host organisms mentioned above by way of example and the vectors suiting the organisms, such as plasmids, viruses or phages, such as the T7 RNA polymerase/promoter system or vectors with regulatory sequences for phage λ.
The term expression systems preferably means the combination of Escherichia coli and its plasmids and phages and the relevant promoters, and Bacillus and its plasmids and promoters.
Also suitable for advantageous novel expression of SEQ ID No. 1 and SEQ ID No. 3 are other 3' and/or 5' terminal regulatory sequences.
These regulatory sequences are intended to make specific expression of the biotin genes and protein expression possible.
This may mean, for example, depending on the host organism that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.
The regulatory sequences and factors may moreover preferably have a beneficial effect on, and thus increase, biotin gene expression. Thus, it is possible and advantageous to enhance the regulatory elements at the level of transcription by using strong transcription signals such as promoters and/or "enhancers". However, it is also possible besides this to enhance translation by, for example, improving the stability of the mRNA.
"Enhancers" mean, for example, DNA sequences which bring about, is via an improved interaction between RNA polymerase and DNA, an increase in biotin gene expression.
An accentuation of the proteins derived from sequences SEQ ID No. 1 or SEQ ID No. 3 and their enzyme activity can be achieved, for example by comparison with the initial enzymes, by modifying the corresponding gene sequences or the sequences of its homologs by classical mutagenesis such as UV irradiation or treatment with chemical mutagens and/or by specific mutagenesis such as site-directed mutagenesis, deletion(s), insertion(s) and/or substitution(s). Increased enzyme activity can also be achieved, besides the gene amplification described, by eliminating factors which repress enzyme biosynthesis and/or by synthesizing active, not inactive, enzymes.
The novel process advantageously increases the conversion of DTB into biotin and thus the biotin productivity overall via the biotin genes having the sequences SEQ ID No. 1 or SEQ ID No. 3 which have been cloned into the organisms via vectors and/or into the chromosome.
In the novel process, the microorganisms comprising SEQ ID No. 1 and/or SEQ ID No. 3 are cultured in a medium which allows these organisms to grow. This medium may be a synthetic or a natural medium. Media known to the skilled worker and depending on the organism are used. The media used for growing the microorganisms comprise a source of carbon, a source of nitrogen, inorganic salts and, where appropriate, small amounts of vitamins and trace elements.
Examples of advantageous sources of carbon are sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose, complex sources of sugars, such as molasses, sugar phosphates such as fructose 1,6-bisphosphate, sugar alcohols such as mannitol, polyols such as glycerol, alcohols such as methanol or ethanol, carboxylic acids such as citric acid, lactic acid or acetic acid, fats such as soybean oil or rapeseed oil, amino acids such as glutamic acid or aspartic acid, or amino sugars, which can also be used as source of nitrogen.
Advantageous sources of nitrogen are organic or inorganic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts such as NH4Cl or (NH4)2SO4, nitrates, urea or complex sources of nitrogen, such as corn steep liquor, brewer's yeast autolysate, soybean flour, wheat gluten, yeast extract, meat extract, casein hydrolysate, yeast or potato protein, which may often also serve as source of nitrogen.
Examples of inorganic salts are the salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper and iron. The anion in these salts which should be particularly mentioned is the chloride, sulfate and phosphate ion. An important factor in enhancing the productivity in the novel process is the addition of Fe2+ or Fe3+ salts and/or potassium salts to the production medium.
Other growth factors are added to the nutrient medium where appropriate, such as vitamins or growth promoters such as riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methionine or lysine, carboxylic acids such as citric acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol.
It is possible, where appropriate, to add antibiotics to the medium to stabilize the vectors with the biotin genes in the cells.
The mixing ratio of said nutrients depends on the mode of fermentation and is established in the individual case. It is possible for the medium components all to be present at the start of the fermentation after they have been, if necessary, sterilized separately or sterilized together, or else to be added during the fermentation as required.
The cultivation conditions are established so that the organisms grow optimally and so that the best possible yields are obtained. Preferred cultivation temperatures are from 15°C C. to 40°C C.
Temperatures from 25°C C. to 37°C C. are particularly advantageous. The pH is preferably kept in a range from 3 to 9. The pH is particularly advantageously from 5 to 8. An incubation time of from 8 to 240 hours, preferably from 8 to 120 hours, is generally sufficient. During this time, the maximum amount of biotin accumulates in the medium and/or is available after disruption of the cells.
The novel process for preparing biotin can be carried out continuously or batchwise. If whole plants are regenerated from the plant cells transformed with the biotin genes, these plants can be grown and propagated quite normally in accordance with the novel process.
Based on the consideration that the conversion of DTB into biotin might involve, apart from BioB and the other known cofactors, possibly an Fe-S cluster-regenerating enzyme, an attempt was made to identify and clone such a hypothetical gene.
NifS genes are able to regenerate sulfur atoms in an Fe-S cluster of proteins involved in nitrogen fixation (Zheng et al., Proc. Natl. Acad. Sci. USA 90, 1993: 2754-2758 and Biochemistry 33, 1994: 4714-4720). It was possible by comparing all known proteins of the NifS class in the Swiss-Prot and PIR databanks using the Lasergene program package (DNA-Star Inc.) in the megalign mode to identify a strictly conserved region of these proteins having the amino acid sequence HK(I,L)xGPxG (x corresponds to amino acids whose conservation is low or not retained). The fact that there is complete conservation of this sequence indicates that these amino acids (=Aa) are important in the functioning of these proteins. These conserved amino acids are referred to as motif I hereinafter.
The Aa motif described in this way was used as comparison sequence for further analysis of the Swiss-Prot/PIR release 93 or 94 databank to search for proteins or ORFs (=open reading frames) in which this NifS functional sequence is completely conserved. The program employed for the sequence analysis was the Geneman program from the DNA-Star package. The analysis parameters were fixed as follows: consensus sequence menu with 80% conservation of the motif.
This search resulted in the finding that, besides proteins which have already been identified as NifS-homologous proteins, there are other proteins or ORFs which have this sequence motif. Among the other sequences present in the databank, it was possible to identify an open reading frame from E. coli which has significant conservation of the consensus sequence and is, as our studies have shown, involved in biotin synthesis. This open reading frame, which is referred to as ECU29581--24 (=SEQ ID No. 1=ORF401), codes for a hypothetical protein of 401 Aa which is derived from this sequence. It emerged that this sequence had been sequenced as part of the E. coli genome sequencing by F. Blattner and co-workers (DNA-Research 1996), without its function having been recognized. This sequence (SEQ ID No. 1) is referred to as BioS1 hereinafter.
Comparison of the protein sequence of BioS1 with that of NifS from A. vinelandii (program: DNA-Star "megalign" in mode: pairwise Lipman-Pearson alignment, analysis parameters: k-tuple 2, gap penalty 4, gap length penalty 12) shows that ECU29581--24 also has a homology of 27.6% with NifS from A. vinelandii in other regions of the sequence over a range of 218 Aa. The homology with the protein identified as NifS from Rhodococcus capsulatus is 25.3% over a range of 376 Aa.
It was possible to identify further sequences having homologies with ECU29581--24 in the SwissProt/PIR databank (Geneman program/sequence similarity mode; default settings). The greatest similarity found to the ECU29581--24 sequence is shown by a translated ORF (=open reading frame) from H. influenzae (databank name HIU00082--62). A sequence homology of 45.5% was found for BioS1 and HIU00082--62 over the entire length of the two proteins. The sequence similarity or the homology of the two proteins is thus significantly greater than between ECU29581--24 (=BioS1) and NifS from R. capsulatus or A. vinelandii. The protein is thus presumably the H. influenzae homolog to BioS1.
Fleischmann et al. (Science. 269, 1995: 496-512) found not only ORF HIU00082--62 but also another ORF (HIU00072--10) in H. influenzae on the basis of its similarity to the NifS sequence.
It was concluded on the basis of this description by Fleischmann et al. that another NifS-like gene, besides bioS1, also exists in E. coli. This hypothetical gene has been called bioS2.
1. Construction of the Vectors pHS1 and pHS2:
The plasmids pHS1 and pHS2 consist of various cassettes which carry an origin of replication, a resistance cassette, a promoter, a cloning site and terminators. The plasmids were assembled from various DNA fragments. The DNA fragments necessary for this were prepared by PCRs with various plasmids as templates.
a.) Preparation of the Cassette with an Origin of Replication:
In order to provide the origin of replication from a P15A replicon-containing plasmid as clonable cassette, a DNA fragment with a length of 919 bases was isolated from the plasmid by a PCR with the plasmid pRep4 (Quiagen) with the oligonucleotides P15A,1 (5'-GGCCCCTAGGGGATATATTCCGCTTCCTCGC-3') (SEQ ID NO:13) and P15A,2 (5'-GGCCACTAGTAACAACTTATATCGTATGGGG-3') (SEQ ID NO:14). The fragment was cut with the restriction enzymes AvrII and SpeI in a suitable buffer.
PCR Conditions:
2.5 U of Taq polymerase and 15 pmol of the oligonucleotides were used in 100 μl of solution to isolate the replication cassette from the plasmid pRep4. The oligonucleotides were annealed at 50°C C. Elongation took place at 72°C C. for 1 min over 30 cycles.
b.) Preparation of the Kanamycin Resistance Cassette:
In order to provide a kanamycin-resistance cassette as clonable cassette, a DNA fragment with a length of 952 bases was isolated by a PCR from a plasmid containing the kanamycin-resistance cassette (pRep4, Quiagen) with the oligonucleotides Kan-R,1 (5'-GGCCGAGCTCTCGAACCCCAGAGTCCCGCT-3') (SEQ ID NO:15) and Kan-R,2 (5'-GGCCGACGTCGGAATTGCCAGCTGGGGCGC-3') (SEQ ID NO:16). The fragment was cut with AatII and SacI in a suitable buffer.
PCR Conditions:
2.5 U of Taq polymerase and 15 pmol of the oligonucleotides were used in 100 μl of solution to isolate the kanamycin resistance cassette from the plasmid pRep4. The oligonucleotides were annealed at 50°C C. Elongation took place at 72°C C. for 1 min over 30 cycles.
c.) Preparation of the Termination Regions:
In order to provide the terminator T0 from phage lambda as clonable cassette, a DNA fragment with a length of 120 bases was isolated by a PCR with the plasmid pDS12-luzi (Schroder H. et al., EMBO Journal. 12, 11, 1993: 4137-4144) as template with the oligonucleotides T0,1 (5'-GGCCGAGCTCGCTTGGACTCCTGTTGATAG-3') (SEQ ID NO:17) and T0,2 (5'-GGCCACTAGTGCTTGGATTCTCACCAATAAAAAACGCCC-3') (SEQ ID NO:18). The fragment was cut by the enzymes SpeI and SacI in a suitable buffer.
Template for T0: pDS12-luzi
2.5 U of Taq polymerase and 15 pmol of the oligonucleotides were used in 100 μl of solution to isolate the termination region from the plasmid pDS12-luzi. The oligonucleotides were annealed at 50°C C. Elongation took place at 72°C C. for 0.5 min over 30 cycles. Then a fragment 120 bp in size was isolated and purified. This fragment was digested with 20 U each of SpeI and SacI.
In order to provide the terminator T1 from the rrnB operon as clonable cassette, a DNA fragment with a length of 120 bp was isolated by a PCR with the plasmid pDS12-luzi (Schroder et al., see above) as template and with the aid of the oligonucleotides T1,1 (5'-GGCCCCTAGGTCTAGGGCGGCGGATTTGTCC-3') (SEQ ID NO:19) and T1,2 (5'-GGCCTCTAGAGGCATCAAATAAAACGAAAGGC-3') (SEQ ID NO:20). The fragment was cut by the enzymes XbaI and AvrII in a suitable buffer.
Template for T1: pDS12-luzi
2.5 U of Taq polymerase and 15 pmol of the oligonucleotides were used in 100 μl of solution to isolate the termination region from the plasmid pDS12-luzi. The oligonucleotides were annealed at 50°C C. Elongation took place at 72°C C. for 0.5 min over 30 cycles. Then a fragment 120 bp in size was isolated and purified. This fragment was digested with 20 U each of XbaI and AvrII.
d.) Preparation of the Promoters for pHS1 and pHS2:
The oligonucleotides PPHS1,1 (5'-TCGAGATAGCATTTTTATCCATAAGATTAGCCGATCCTAAGGTTTACAATTGTGAGCGCTC CAATTATGATAGATTCAATTGTGAGCGGATAACAATTTCACACACGCTAGCGGTAC-3') (SEQ ID NO:21) and PPHS1,2 (5'-CGCTAGCGTGTGTGAAATTGTTATCCGCTCACAATTGAATCTATCATAATTGTGAGCGCTC ACAATTGTAAACCTTAGGATCGGCTAATCTTATGGATAAAAATGCTATC-3') (SEQ ID NO:22) and PPHS2,1 (5'-AATTCTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGACATC ACCAGGACGCACTGACCG-3') (SEQ ID NO:23) and PPHS2,2 (5'-AATTCGGTCAGTGCGTCCTGGTGATGTCTCAGTATCTCTATCACTGATAGGGATGTCAATC TCTATCACTGATAGGGAGG-3') (SEQ ID NO:24) were prepared by chemical synthesis. The oligonucleotides PPHS1,1 and PPHS1,2 and PPHS2,1 and PPHS2,2, were respectively mixed in a concentration of 1 μg/μl, incubated at 95°C C. for 5 min and then cooled slowly. The annealed oligonucleotides were employed in a concentration of 10 ng/μl in the ligation. Oligonucleotides PPHS1,1 and PPHS1,2 formed the promoter for the plasmid pHS1, and oligonucleotides PPHS2,1 and PPHS2,2 formed that for the plasmid pHS2.
e.) Preparation of the Cloning Site:
To construct the cloning site, the two oligonucleotides PMCS1,1 (5'-GTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCC CGGGGGATCCCATGGTA-3') (SEQ ID NO:25) and PMCS1,2 (5'-ACGCGTACCATGGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCG ACCTCGAGGGGGGGCCCGGTACC-3') (SEQ ID NO:26) were synthesized. The two oligonucleotides were mixed in a concentration of 1 μg/μl, incubated at 95°C C. for 5 min and then cooled slowly. The annealed oligonucleotides were employed in a concentration of 10 ng/μl in the ligation.
f.) Procedure for Cloning pHS1 and pHS2
Starting from pDS12 luci, the ampR cassette of the plasmid was cut out by SacI/AatII digestion and replaced by a corresponding SacI/AatII fragment which contains the kanamycin resistance cassette. The vector obtained after transformation and isolation of positive clones underwent SpeI/SacI digestion, and the PCR-amplified terminator T0 was inserted as SpeI/SacI fragment downstream of the kanamycin resistance cassette. The vector obtained after transformation and isolation of positive clones was digested with XbaI/AvrII, and the PCR-amplified terminator T1 was inserted as xbaI/AvrII fragment. The resulting vector was digested with XhoI/EcoRI and ligated to the respectively annealed promoter oligonucleotides (PPHS1,1 and PPHS1,2, and PPHS2,1 and PPHS2,2). The resulting vector underwent XbaI digestion and was filled in using the Klenow fragment and then, after KpnI digestion, the vector band without the luziferase fragment was isolated. Two further annealed oligonucleotides (PMCS1,1 PMCS1,2) which contain the cloning site were ligated to the vectors digested in this way.
2. Cloning of bioS1 (ECU29581--24, SEQ ID No. 1):
The gene which codes for BioS1 was amplified from the chromosome of E. coli by a PCR, provided with optimized translation signals, and cloned into a vector which makes overexpression of the gene in E. coli strains possible.
a.) Development of Oligonucleotides for Amplifying the bioS1 Gene from the E. coli Chromosome:
BioS1 is to be amplified as expression cassette consisting of a ribosome binding site, the start codon of the coding sequence and the stop codon between two recognition sites for restriction enzymes. The MluI recognition sequence was chosen for both restriction cleavage sites. The bioS1 gene was cloned with the aid of the oligonucleotides PbioS1,1 (5, -CGCACGCGTGAGGAGTACCATGAACGT-3') (SEQ ID NO:27) and PbioS1,2 (5'-CGCACGCGTTTAATCCACCAATAATT-3') (SEQ ID NO:28).
PCR Procedure:
Conditions:
0,5 μg of chromosomal DNA from E. coli W3110 was used as template. The oligonucleotides PbioS1,1 and PbioS1,2 were each employed in a concentration of 15 pM. The concentration of dNTPs was 200 μM. 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in the manufacturer's reaction buffer were used as polymerase. The volume for the PCR was 100 μl.
Amplification Conditions:
The DNA was denatured at 94°C C. for 2 min. The oligonucleotides were then annealed at 55°C C. for 30 sec. Elongation took place at 72°C C. for 45 sec. The PCR was carried over 30 cycles.
The resulting DNA product with a size of 1200 bp was purified and digested by MluI in a suitable buffer.
5 μg of the vector pHS1 were digested by MluI and dephosphorylated by shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After denaturation of the SAP, the vector and fragment were ligated in a molar ratio of 1:3 by the rapid DNA ligation kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into the strain E. coli XL1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the bioS1 fragment in pHS1 was determined by restriction digestion and sequencing. The resulting construct was called pHS1 bioS1 (FIG. 1). The sequence of pHS1 bioS1 is to be found in SEQ ID No. 5. The derived amino acid sequence of bioS1 in the vector is to be found in SEQ ID No. 6. 2 μg of the vector pHS1 bioS1 were digested with MluI, and the gene bioS1 was isolated by an agarose gel. The vector pHS2 was digested with MluI, dephosphorylated with SAP and attached to the fragment bioS1 in a joint ligation. The ligation mixture was transformed into XL1-blue, and positive clones in the correct orientation were identified by plasmid isolation and restriction digestion. The resulting vector was called pHS2 bioS1 (FIG. 2). The sequence of pHS2 bioS1 is to be found in SEQ ID No. 9. The derived amino acid sequence of bioS1 in the vector is to be found in SEQ ID No. 10.
3. Cloning of bioS2 (SEQ ID No. 3):
The way of cloning into E. coli further genes which code for gene products which are involved in the assembly of Fe-S clusters was as follows:
A sequence comparison (Megalign program, Clustal mode) of Aa sequences of proteins in the SwissProt/PIR databank with high homology (>40%) with the NifS protein from A. vinelandii showed that, besides the sequence motif I which has been described above, the N terminus of these proteins also shows significant conservation. The Aa sequence MIYLDNXATT (SEQ ID NO:29) was identified as a typical N-terminal conserved sequence of proteins of the NifS family and was called motif II a.
Analysis of the SwissProt/PIR databank for more than 80% conservation of this sequence showed another protein in E. coli. 11 Aa in the N-terminal sequence of this protein are known from Edman degradation (databank name: UP06--Ecoli). This protein was regarded as hypothetical NifS homolog and is referred to as BioS2, in analogy to BioS1, hereinafter.
The way of cloning and sequencing the gene for the protein BioS2 was as follows. Starting from the protein sequence HIU00072--10, on the one hand the conserved amino acid motif I, and on the other hand the abovementioned Aa sequence of UP06--Ecoli, were used to prepare degenerate oligonucleotides which are able to amplify a fragment of the bioS2 gene. For this purpose, the two Aa motifs HIU00072--10 (motif I) and UP06--Ecoli (motif II b, MKLPIYLDYSAT (SEQ ID NO:30)) were reverse translated into the corresponding DNA sequences. In this way, the degenerate oligonucleotide PbioS2,1 (5'-ATGAARYTNCCNATHTAYYTNGAYTAYWSNGCNAC-3') (SEQ ID NO:31) was synthesized from motif II, and the degenerate oligonucleotide PbioS2,2 (5'-cccaghggrccrtgcagyttrtgrccrga-3') (SEQ ID NO:32) was synthesized from motif I.
PCR Procedure:
Chromosomal DNA from E. coli W3110 was used as template. 0.5 μg each of the oligonucleotides PbioS2,1 and PbioS2,2 were reacted in each case with 15 pmol of nucleotide mix, 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in the manufacturer's reaction buffer. The volume for the PCR was 100 μl.
Amplification Conditions:
Denaturation took place at 94°C C. for 2 min. Annealing of the oligonucleotides was carried out at 45°C C., and elongation was carried out at 72°C C. for 45 sec. The PCR was carried out over 30 cycles. It was possible by the PCR to amplify three fragments selectively, and one of them had the size of 600 bp expected from the sequence comparisons. This DNA fragment was isolated by agarose gel purification and sequenced with the oligonucleotide PbioS2,2. The resulting DNA sequence was translated in all six possible reading frames. The resulting translated Aa sequences were then compared with the translated Aa sequence of HIU00072--10 and NifS from A. vinelandii. One of the translated reading frames shows high homology with the Aa sequence of the described ORF HIU00072--10 an d is called bioS2.
The way of determining and cloning the complete DNA sequence of bioS2 (=SEQ ID No. 3) was as follows:
Firstly, a labeled DNA probe homologous with bioS2 was prepared. This took place using the PCR-DIG labeling kit (Boehringer Mannheim). The template used for preparing the DIG-DNA probe was the described PCR product which was prepared by the oligonucleotides PbioS2,1 and PbioS2,2.
The PCR Conditions Were:
Used: 1 μl of the PCR template, 5 μl of nucleotide DIG-dUTP mix, 15 pmol each of oligonucleotide PbioS2,1 and PbioS2,2, buffer from the kit with 1.75 MM MgCl2, 0.75 μl of expand polymerase mix (Boehringer Mannheim).
Amplification Conditions:
Melting of the DNA at 94°C C. for 2 min, melting of the DNA at 94°C C. for 10 sec, annealing at 45°C C. for 30 sec, elongation at 68°C C. for 3.30 min over 10 cycles, melting of the DNA at 94°C C. for 10 sec, annealing at 45°C C. for 30 sec, elongation at 68°C C. for 3.30 min, extension of the elongation for 20 sec per cycle over 20 cycles. Purification of the DIG-labeled fragment by PCR purification kit.
4. Southern Analysis of bioS2 with Chromosomal DNA
In further steps, genomic DNA was digested by restriction enzymes and analyzed by Southern hybridization using the labeled DNA probe.
Chromosomal E. coli DNA W3110 (10 μg) was completely digested with the following restriction enzymes: EcoRI, BamHI, Acc65I, HindIII, SalI. The volume of the mixtures was 50 μl, and the amount of each enzyme was 30 U. The mixtures were incubated for 4 h. The DNA digested in this way was fractionated through a 1% agarose gel in TBE buffer (Sambrook, J. Fritsch, E F. Maniatis, T. 2nd ed. Cold Spring Harbor Laboratory Press., 1989, ISBN 0-87969-373-8) and transferred with the aid of a pressure transfer chamber (Stratagene) to a nylon membrane (Boehringer Mannheim) and covalently fixed to the membrane by UV irradiation (Stratalinker, Stratagene). The hybridization with the DIG-labeled DNA probe took place in DIG Easyhyb buffer (Boehringer Mannheim) at 65°C C. for 15 h. Development of the blot in accordance with the manufacturer's instructions shows hybridization of the BioS2 DIG-DNA probe with bands whose sizes were determined to be about 3-4 kb in the case of Acc65I, EcoRI and HindIII. BamHI-digested DNA showed hybridization with the bioS2 probe with a fragment of a considerably higher molecular weight. 3-4 kb fragments were preferably used for further cloning.
Cloning of bioS2 by Inverse PCR
In the case of the EcoRI digestion, a fragment of about 4 kb which harbors the gene which is sought was identified. The complete gene was then amplified and cloned by the inverse PCR technique. In the first step of the inverse PCR, chromosomal E. coli DNA was completely digested by EcoRI. In the second step, the EcoRI-digested DNA was then covalently ligated at low DNA concentrations (about 20 ng/ml) from the previously described restriction digestion by ligase under conditions under which, viewed statistically, there is intramolecular linkage. In the third step, a PCR was then carried out employing oligonucleotides whose sequence is specific for the target gene which is sought.
Specifically amplified DNA segments can be identified on the basis of their size, which emerges from the size of the restriction fragment from the Southern analysis and from the localization of the oligonucleotides in the known section of the gene. Fragments identified in this way are then cloned into a suitable vector such as pBS SK Bluescript/pCR Script (Stratagene), and sequenced.
Experimental Procedure:
1 μg of chromosomal DNA from the strain W3110 was completely digested by 15 U of EcoRI (Boehringer Mannheim) in a volume of 50 μl. The completeness of digestion was checked by loading 30 μl onto an agarose gel. Fragments from this digestion of chromosomal DNA (10 μl of the digest=200 ng) were incubated in a volume of 100 μl together with 10 μl of ligation buffer and 2 U of T4 ligase (Boehringer Mannheim) at 15°C C. for 15 h (intramolecular ligation reaction). After the ligation reaction, the T4 ligase was inactivated by incubation at 65°C C. for 20 min. 5 μl of this ligation mixture were used as template for a PCR. The primers PbioS2,3 (5'-GCGTGGGTAAACTGCCTATCGACCTGAGCC-3') (SEQ ID NO:33) and PbioS2,4 (5'-CTACGCTTCCTTCAGCCTGCCAGCCGAAA-3') (SEQ ID NO:34) were synthesized starting from the sequence of bioS2.
Oligonucleotide PbioS2,3 hybridizes on the 5' side of bioS2 and results in the elongation of amplification of the coding sequence taking place on the 5' side on the complementary strand in the 3' direction.
Oligonucleotide PbioS2,4 hybridizes on the 3' side of bioS2 and results in the elongation of amplification of the coding sequence taking place on the 3' side on the coding strand in the 3' direction.
Used: 5 μl of the ligation mixture, 1.75 μl of deoxynucleotide mixture (350 μmol, Boehringer Mannheim), 15 pmol of each oligonucleotide PbioS2,5 and PbioS2,6, buffer 1 from the kit with 1.75 mM MgCl2, 0.75 μl of expand polymerase mix.
Conditions for the amplification of the ligated E. coli DNA with primer PbioS2,3/PbioS2,4. Expand kit (Boehringer Mannheim) melting of the DNA at 94°C C. for 2 min, melting of the DNA at 94°C C. for 10 sec, annealing at 61°C C. for 30 sec, elongation at 68°C C. for 3.30 min over 10 cycles, melting of the DNA at 94°C C. for 10 sec, annealing at 61°C C. for 30 sec, elongation at 68°C C. for 3.30 min, extension of the elongation for 20 sec per cycle over 20 cycles.
The described amplification resulted in a PCR product of about 3 kb. This DNA fragment showed pronounced Southern hybridization with the bioS2-DIG-DNA probe described above under stringent conditions.
It was assumed that this DNA fragment comprises DNA sequences which are highly homologous with bioS2. This DNA fragment was therefore cloned into a vector in order to characterize it further and sequence it. Using the pCR Script kit (Stratagene), the DNA fragment was first treated with Pfu polymerase in accordance with the manufacturer's instructions, and then ligated into the vector pCR Script. The ligation mixture was transformed into XL1-blue cells (Stratagene) and plated out on LB-Amp. A positive clone which harbored a fragment was identified by minipreparation analysis. Sequencing revealed the complete sequence depicted in SEQ ID No. 3 (=bioS2).
BioS2 was then amplified and cloned as expression cassette as for bioS1. For this purpose, an MluI recognition site and an optimized Shine-Dalgarno sequence were added on the 5' side of the gene, and an MluI recognition site was added on the 3' side of the gene, by PCR with the oligonucleotides PbioS2,5 (5'-CATGACGCGTAAAGAGGAGAAATTAACTATGAAATTACCGATTTATTTGG-3') (SEQ ID NO:35) and PbioS2,6 (5'-GCGACGCGTGATTAATGATGAGCCCAT-3') (SEQ ID NO:36).
PCR Procedure:
0.5 μg of chromosomal DNA from W3110 was employed as template. The oligonucleotides PbioS2,5 and PbioS2,6 were each employed in a concentration of 15 pM. The concentration of dNTPs was 200 μM. 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) were employed as polymerase in the manufacturer's reaction buffer. The volume for the PCR was 100 μl.
Amplification Conditions:
Denaturation at 94°C C. for 2 sec, annealing of the oligonucleotides at 55°C C. for 30 sec, elongation at 72°C C. for 45 sec. The PCR was carried out over 30 cycles.
The resulting DNA product with the correct size of about 1200 bp was purified by the PCR purification kit (Boehringer Mannheim) and digested by MluI in a suitable. buffer.
5 μg of the vector pHS2 were digested by MluI and dephosphorylated by shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After denaturation of the SAP, the vector and fragment were ligated in a molar ratio of 1:3 by the rapid DNA ligation kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into the strain XL1-blue. Positive clones were identified by plasmid preparation and restriction analysis. Correct orientation of the bioS2 fragment in pHS2 was determined by restriction digestion and sequencing. The vector was called pHS2 bioS2 (FIG. 3). The sequence of pHS2 bioS2 is to be found in SEQ ID No. 11. The derived amino acid sequence of bioS2 in the vector is to be found in SEQ ID No. 12. Cloning of bioS2 into the vector pHS1 was carried out in a similar way. The sequence of pHS1 bioS2 is to be found in SEQ ID No. 7 (FIG. 4). The derived amino acid sequence of bioS2 in the vector is to be found in SEQ ID No. 8.
5. Construction of the plasmid pHBbio14
The Bio operon was cloned in vivo by a transducing lambda phage. Lambda bio+phages were selected by transduction of an E. coli bio-negative strain to bio+. The isolated bio+ transducing lambda phage was propagated, and the lambda DNA was purified. This was followed by excision of an 8.7 kb EcoRI/HindIII fragment with the complete biotin operon out of the lambda phage DNA and ligation of the fragment into pBR322 which had been cut with EcoRI/HindIII. Positive clones were identified by plasmid preparation and restriction analysis.
Deletion of a 1.2 kb 3' Fragment of bioD
Unnecessary gene sequences on the 3' side of the bioD gene were deleted. For this purpose, an EcoRI cleavage site was introduced behind the bioD stop codon by PCR. The oligonucleotides Pbio1,1 (5'-AATAAGGAATTCTTATGTACTTTCCGGTTGCCG-3') (SEQ ID NO:37) and Pbio1,2 (5'-AACAGCAGCCTGCAGCTGGATTA-3') (SEQ ID NO:38) for this PCR were developed according to the operon sequence of Otsuka et al. (J. Biol. Chem. 263, 1988: 19577-85).
PCR Conditions:
2,5 U of Taq polymerase (Perkin Elmer) and 15 pmol of each primer were reacted in a volume of 100 μl. The annealing was carried out at 50°C C. and the elongation at 72°C C. for 1 min over 30 cycles. A 488 bp fragment was isolated and purified on an agarose gel.
Digestion of the resulting fragment with EcoRI/PstI. Digestion of pHBbio1 with EcoRI/PstI. Isolation of a 9.5 kb fragment.
The 9.5 kb fragment was ligated to the 488 bp fragment and transformed into XL1-blue cells. Resulting clones were analyzed by plasmid preparation and by restriction analysis of the plasmid DNA using the enzymes EcoRI and HindIII, and positive clones which harbored a 5.9 kb fragment were identified. A clone was isolated and was called pHBbio2. Plasmid DNA was obtained from this clone. 5 μg of pHBbio2 were digested with EcoRI/HindIII, and the 5.9 kb fragment which contained the complete biotin biosynthesis genes was isolated.
5 μg of the plasmid pAT153 were digested with EcoRI and HindIII. The resulting 5.9 kb fragment with the biotin biosynthesis genes was ligated to the digested vector pAT153 and transformed into XL1-blue. Resulting clones were analyzed by plasmid preparation and by restriction analysis of the plasmid DNA with the enzymes EcoRI and HindIII. Positive clones were identified, and a clone was isolated and called pHBbio14.
6. Increasing Biotin Productivity by Overexpression of bioS1
Strain BM4092 (Barker and Campbell) was transduced to recA- by a P1 transduction using a P1 lysate which had grown on a recA::Tn10-harboring strain. Success of the transduction was detected by increased UV sensitivity of the positive transductants. The resulting strain LU8091 was then transformed with the plasmid pHBbio14 by the CaCl2 method and was cultured on LB-ampicillin 100 μg/ml. One clone was isolated, and this was transformed with each of the plasmids pHS1 bioS1 and pHS2 bioS2 by the CaCl2 method and selected on LB agar, ampicillin 100 μg/ml and kanamycin 25 μg/ml.
One colony of each of the transformants was inoculated in a DYT culture with the appropriate antibiotic and incubated for 12 h. The overnight culture was employed for inoculation of a 10 ml culture in TB medium (Sambrook, J. Fritsch, E F. Maniatis, T. 2nd ed. Cold Spring Harbor Laboratory Press., 1989 ISBN 0-87969-373-8) with the appropriate antibiotics, and was cultured for 24 h. After completion of growth, the cells were removed from the culture supernatant by centrifugation, and the biotin and dethiobiotin concentrations were determined by an ELISA with streptavidin and avidin in the supernatant. The results of this determination are to be found in Table I.
| TABLE I | ||||
| Determination of the biotin and dethiobiotin | ||||
| concentrations | ||||
| Strain | Plasmid I | Plasmid II | Biotin mg/l | Dethiobiotin mg/l |
| Lu8091 | pHBbio14 | 9.4 | 45.6 | |
| Lu8091 | pHBbio14 | pHS1 bioS1 | 15.3 | 19.7 |
| Lu8091 | pHBbio14 | pHS2 bioS1 | 19.2 | 15.8 |
| TABLE I | ||||
| Determination of the biotin and dethiobiotin | ||||
| concentrations | ||||
| Strain | Plasmid I | Plasmid II | Biotin mg/l | Dethiobiotin mg/l |
| Lu8091 | pHBbio14 | 9.4 | 45.6 | |
| Lu8091 | pHBbio14 | pHS1 bioS1 | 15.3 | 19.7 |
| Lu8091 | pHBbio14 | pHS2 bioS1 | 19.2 | 15.8 |
Hauer, Bernhard, Schröder, Hartwig
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